It is proposed to study the structural aspects of protein allosterism in human hemoglobin and regulatory enzymes by exploiting the power of the X-ray crystallographic method to elucidate protein conformation on the atomic level. A long-term goal of this research program will be the determination of the 3-dimensional structure of one or more regulatory enzymes. This work will entail the crystallization of selected enzymes in forms suitable for high resolution X-ray study, followed by a search for isomorphous heavy-atom derivatives, and finally the calculation and analysis of electron density maps. However, by using the method of difference electron density analysis, the utility of the crystallographic method can be extended beyond the initial goal of solving the structure of a protein. The major portion of the proposed research will make use of this technique to study nonheme-ligand binding to hemoglobin. Specifically: 1) A new method for directly determining the approximate pK values of titratable residues is described and shown to be feasible. It is planned to use this approach to identify the residues responsible for the unassigned portion of the alkaline Bohr effect. 2) The interaction of CO2 with deoxyhemoglobin will be studied under a variety of conditions. 3) The effect of 2,3-diphosphoglycerate binding on the tertiary structure of deoxyhemoglobin will be studied in detail at high resolution. 4) The structural reasons for the ability of inositol hexaphosphate to rejuvenate the functional properties of carboxypeptidase A - digested hemoglobin will be sought. 5) The binding of drugs of potential value to the treatment of sickle cell disease will be studied in order to determine the structural basis of their action. 6) The changes in structure of a number of hemoglobin mutants will be determined in order to correlate changes in structures with changes in function. In addition, it is proposed to elucidate the structure of hemoglobin H (Beta4) and to compare it with the structure of hemoglobin A (alpha2 Beta2) in an effort to identify the intersubunit contacts which are vital for the proper functioning of hemoglobin A.